The primary objective of the proposed research is to determine how the balance between the protective and genome delivery functions of the viral capsid influences viral pathogenesis. The goal will be attained using the tractable mammalian reovirus system as an experimental model. In its native state, the ?1 protein of the reovirus outer capsid confers stability to the viral particle. During entry into cells, ?1 is exposed and cleaved to generate ?1N, ?, and ? fragments. These ?1-derived peptides are buried and remain associated with the virus particle. Upon interaction with host membranes, the ?1 protein undergoes a dramatic conformational rearrangement to expose and release ?1N and ?. The released peptides form pores in target membranes that allow delivery of the ~70 nm viral inner capsid (core) across membranes. Three integrated aims are proposed to define how the mutually antagonistic, protective and genome delivery functions of ?1 are balanced, and how this balance modulates viral disease. In Aim 1, how the reovirus capsid is destabilized to promote conformational changes required for cell entry will be determined. Viral determinants that modulate capsid stability and conformational flexibility will be identified. The mechanisms by which host proteases promote conformational changes in the viral capsid will be defined. A subnanometer structure of the conformationally-altered reovirus particle will be solved by cryo-electron microscopy (cryo-EM). In Aim 2, how membranes are breached by ?1 will be elucidated. The minimal number of pore-forming peptides needed to initiate infection will be determined. Features within ?1N and ? that allow these peptides to form pores will be defined. Determinants within the capsid that control the shape and size of the pore formed by reovirus during cell entry will be identified. Structures of viral entry intermediates in association with model membranes will be determined by cryo-EM. In Aim 3, how functions of ?1 in maintaining capsid stability and allowing genome delivery influence viral pathogenesis will be determined. The capacity of ?1 mutant viruses with altered stability, conformational flexibility, or genome delivery efficiency to replicate at initial sites of infection, disseminate to secondary sites of infection, and replicate at secondary sites will be evaluated in a newborn mouse model. The virulence of ?1 mutant viruses will be compared. The effect of capsid stability on transmission between animals will also be determined. Successful completion of these goals will define how different functions of the viral capsid are regulated, provide unprecedented snapshots of changes occurring in the viral capsid during its transit across the membrane, and identify a relationship between viral capsid properties, tissue tropism, and viral disease. This work will help identify critical control points in the conserved cell entry pathway of nonenveloped viruses that could serve as potential targets for antiviral therapeutics. In addition, results of these studies could foster the development of a more efficacious reovirus oncolytic.